严重烧伤患者休克期肝素结合蛋白的变化及其对人脐静脉血管内皮细胞和中性粒细胞的影响  

Changes of heparin-binding protein in severe burn patients during shock stage and its effects on human umbilical vein endothelial cells and neutrophils

作　　者：戚欣欣 刘璐 杨云稀 黄佳敏 孙炳伟 Qi Xinxin;Liu Lu;Yang Yunxi;Huang Jiamin;Sun Bingwei(School of Medicine,Jiangsu University,Zhenjiang 212001,China;Department of Burns and Plastic Surgery,Affiliated Suzhou Hospital of Nanjing Medical University,Suzhou 215002,China)

机构地区：[1]江苏大学医学院,镇江212001 [2]南京医科大学附属苏州医院烧伤整形科,苏州215002

出　　处：《中华烧伤与创面修复杂志》2022年第2期147-155,共9页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金面上项目(81772135,82072217);江苏省自然科学基金(BK20201178)。

摘　　要：目的探讨严重烧伤患者休克期肝素结合蛋白(HBP)的变化及其在体外对人脐静脉血管内皮细胞(HUVEC)和中性粒细胞的影响。方法采用前瞻性观察性研究与实验研究方法。将2020年8—11月南京医科大学附属苏州医院烧伤整形科收治的符合入选标准的20例严重烧伤患者纳入严重烧伤组[男12例、女8例,年龄为44.5(31.0,58.0)岁],同期招募该单位体检中心体检结果正常的20名健康志愿者纳入健康对照组[男13例、女7例,年龄为39.5(26.0,53.0)岁]。采用酶联免疫吸附测定(ELISA)法检测健康对照组志愿者血浆与严重烧伤组患者伤后48 h内血浆中HBP和组织金属蛋白酶抑制物1(TIMP-1)蛋白表达水平,分别对2组血浆中HBP和TIMP-1蛋白表达的相关性进行Pearson相关分析。取第4代对数生长期HUVEC进行实验,将细胞按随机数字表法(分组方法下同)分为常规培养的正常对照组(处理下同)与进行相应处理的重组HBP(rHBP)处理12 h组、rHBP处理24 h组、rHBP处理48 h组,采用实时荧光定量反转录PCR法检测细胞中TIMP-1的mRNA表达;将细胞分为正常对照组与进行相应处理的rHBP处理48 h组,采用蛋白质印迹法检测细胞中TIMP-1蛋白表达;将细胞分为正常对照组与加入相应试剂处理的单纯rHBP组、单纯抑蛋白酶多肽组、rHBP+抑蛋白酶多肽组(rHBP终物质的量浓度为200 nmol/L,抑蛋白酶多肽终质量浓度为20μg/mL),培养48 h,采用ELISA法检测细胞培养上清液中TIMP-1蛋白表达水平。采用免疫磁珠分选法从前述10名健康志愿者外周静脉血中分离中性粒细胞,将细胞分为正常对照组与加入相应试剂处理的单纯重组TIMP-1(rTIMP-1)组、单纯佛波酯组、rTIMP-1+佛波酯组(rTIMP-1终质量浓度为500 ng/mL,佛波酯终物质的量浓度为10 nmol/L),培养1 h,采用免疫荧光法观测细胞中CD63蛋白表达,采用流式细胞术检测细胞中CD63蛋白阳性表达率,采用ELISA法检测细胞培养上清液中HBObjective To investigate the changes of heparin-binding protein(HBP)in severe burn patients during shock stage and its effects on human umbilical vein endothelial cells(HUVECs)and neutrophils in vitro.Methods Prospective observational and experimental research methods were used.Twenty severe burn patients who met the inclusion criteria and were admitted to the Department of Burns and Plastic Surgery of Affiliated Suzhou Hospital of Nanjing Medical University from August to November 2020 were included in severe burn group(12 males and 8 females,aged 44.5(31.0,58.0)years).During the same period,20 healthy volunteers with normal physical examination results in the unit's Physical Examination Center were recruited into healthy control group(13 males and 7 females,aged 39.5(26.0,53.0)years).Enzyme-linked immunosorbent assay(ELISA)method was used to detect the protein expression levels of HBP and tissue inhibitor of metalloproteinase 1(TIMP-1)in plasma of patients within 48 hours after injury in severe burn group and in plasma of volunteers in healthy control group.The correlation between protein expression of HBP and that of TIMP-1 in the plasma in the two groups was analyzed by Pearson correlation analysis.The fourth passage of HUVECs in logarithmic growth phase were used for the experiment.The HUVECs were divided into normal control group with routine culture(the same treatment below)and recombinant HBP(rHBP)-treated 12 h group,rHBP-treated 24 h group,and rHBP-treated 48 h group with corresponding treatment according to the random number table(the same grouping method below),and the mRNA expression of TIMP-1 in cells was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction.The HUVECs were divided into normal control group and rHBP-treated 48 h group with corresponding treatment,and the protein expression of TIMP-1 in the cells was detected by Western blotting.The HUVECs were divided into normal control group,rHBP alone group,aprotinin alone group,and rHBP+aprotinin group tr

关 键 词：烧伤 金属蛋白酶1组织抑制剂 人脐静脉内皮细胞 细胞脱颗粒 肝素结合蛋白 中性粒细胞 

分 类 号：R644[医药卫生—外科学]